The Role of Myocyte Extracellular Vesicles in Inducing Apoptosis of Murine Lung Cancer CMT64/61 Cells

Extracellular vehicles (EVs) are a heterogeneous group of particles ranging from 15 nm to 1000 nm in diameter, released by almost every cell type, including tumours. These vesicles contain a complex cargo, including protein, lipid and nucleic acids, which reflect the status of the cell of origin. Increasing evidence suggests that EVs play an important role in intercellular communication, both locally and systemically, by transferring their cargo between cells and inducing phenotypical and functional changes in recipient cells. In cancer, the pro-tumourigenic role of EVs is well established; however, their capability as an anti-tumourigenic agent is still emerging. This study identified a novel anti-tumourigenic role of skeletal muscle (C2C12) derived EVs towards highly metastatic lung carcinoma cells (CMT 64/61). Skeletal muscle is a rare site for malignant metastasis and the mechanism underling the rarity of this phenomenon has remained elusive. The data obtained in this study indicated that myocyte EVs at low concentration, up to 200 μg/ml, exert cytotoxic effects on lung carcinoma cells, whilst having no effect on normal fibroblast cells (NIH 3T3). Myocyte EVs induced morphological changes in carcinoma cell mitochondria, decreased mitochondrial membrane potential leading to increased caspase 3 and 9 activity and apoptosis. A significant % of apoptosis, 34.8%, was exerted by 200 μg/ml of myocyte EVs within 48 h. No significant apoptosis was seen in non-carcinoma cells. Cell cycle analysis revealed that myocyte EVs mediated carcinoma cell proliferation suppression via cell cycle arrest at S phase. Transwell migration assay indicated a dose dependent reduction in carcinoma cell migration towards the microenvironment containing myocyte EVs. To further explore the possible protein cargo that exert above effects, proteomics analysis was conducted on myocyte EVs. Scaffold software identified 29 upregulated proteins in myocyte EVs and interestingly, STRING and KEGG analysis on these proteins identified 3 possible pathways that could exert cytotoxic, apoptotic and cytostatic effects on carcinoma cells. Proteins including Cathepsin L1, Cathepsin B and Cathepsin D, involved in Lysosome and Apoptosis pathway whilst Decorin, Thrombospondin-1 and Cathepsin L1 in Proteoglycan in cancer pathway. While all of these proteins may contribute to the effects observed in carcinoma cells, Decorin (DCN) seemed a promising target due its already known anti-tumourigenic properties. Together these results indicate that skeletal muscle derived EVs function as an anti-tumourigenic agent; hence identifies as a potential therapeutic agent in the treatment of metastatic lung carcinoma.